The present invention relates to conformationally constrained Nxcex1 backbone-cyclized peptide analogs cyclized via novel non-peptidic linkages, to novel Nxcex1,xcfx89-functionalized amino acid building units, to processes for the preparation of these backbone cyclized peptides and building units, to methods for using these peptide analogs and to pharmaceutical compositions containing same.
Peptidomimetics
As a result of major advances in organic chemistry and in molecular biology, many bioactive peptides can now be prepared in quantities sufficient for pharmacological and clinical utilities. Thus in the last few years new methods have been established for the treatment and therapy of illnesses in which peptides have been implicated. However, the use of peptides as drugs is limited by the following factors: a) their low metabolic stability towards proteolysis in the gastrointestinal tract and in serum; b) their poor absorption after oral ingestion, in particular due to their relatively high molecular mass or the lack of specific transport systems or both; c) their rapid excretion through the liver and kidneys; and d) their undesired side effects in non-target organ systems, since peptide receptors can be widely distributed in an organism.
Moreover, with few exceptions, native peptides of small to medium size (less than 30-50 amino acids) exist unordered in dilute aqueous solution in a multitude of conformations in dynamic equilibrium which may lead to lack of receptor selectivity, metabolic susceptibilities and hamper attempts to determine the biologically active conformation. If a peptide has the biologically active conformation per se, i.e., receptor-bound conformation, then an increased affinity toward the receptor is expected, since the decrease in entropy on binding is less than that on the binding of a flexible peptide. It is therefore important to strive for and develop ordered, uniform and biologically active peptides.
In recent years, intensive efforts have been made to develop peptidomimetics or peptide analogs that display more favorable pharmacological properties than their prototype native peptides. The native peptide itself, the pharmacological properties of which have been optimized, generally serves as a lead for the development of these peptidomimetics. However, a major problem in the development of such agents is the discovery of the active region of a biologically active peptide. For instance, frequently only a small number of amino acids (usually four to eight) are responsible for the recognition of a peptide ligand by a receptor. Once this biologically active site is determined a lead structure for development of peptidomimetic can be optimized, for example by molecular modeling programs.
As used herein, a xe2x80x9cpeptidomimeticxe2x80x9d is a compound that, as a ligand of a receptor, can imitate (agonist) or block (antagonist) the biological effect of a peptide at the receptor level. The following factors should be considered to achieve the best possible agonist peptidomimetic a) metabolic stability, b) good bioavailability, c) high receptor affinity and receptor selectivity, and d) minimal side effects.
From the pharmacological and medical viewpoint it is frequently desirable to not only imitate the effect of the peptide at the receptor level (agonist) but also to block the receptor when required (antagonism). The same pharmacological considerations for designing an agonist peptidomimetic mentioned above hold for designing peptide antagonists, but, in addition, their development in the absence of lead structures is more difficult. Even today it is not unequivocally clear which factors are decisive for the agonistic effect and which are for the antagonistic effect.
A generally applicable and successful method recently has been the development of conformationally restricted peptidomimetics that imitate the receptor-bound conformation of the endogenous peptide ligands as closely as possible (Rizo and Gierasch, Ann. Rev. Biochem., 61:387, 1992). Investigations of these types of analogs show them to have increased resistance toward proteases, that is, an increase in metabolic stability, as well as increased selectivity and thereby fewer side effects (Veber and Friedinger, Trends Neurosci., p. 392, 1985).
Once these peptidomimetic compounds with rigid conformations are produced, the most active structures are selected by studying the conformation-activity relationships. Such conformational constraints can involve short range (local) modifications of structure or long range (global) conformational restraints (for review see Giannis and Kolter, Angew. Chem. Int. Ed. Enal. 32:1244, 1993).
Conformationally Constrained Peptides
Bridging between two neighboring amino acids in a peptide leads to a local conformational modification, the flexibility of which is limited in comparison with that of regular dipeptides. Some possibilities for forming such bridges include incorporation of lactams and piperazinones. xcex3-Lactams and xcex4-lactams have been designed to some extent as xe2x80x9cturn mimeticsxe2x80x9d; in several cases the incorporation of such structures into peptides leads to biologically active compounds.
Global restrictions in the conformation of a peptide are possible by limiting the flexibility of the peptide strand through cyclization (Hruby et al., Biochem. J., 268:249, 1990). Not only does cyclization of bioactive peptides improve their metabolic stability and receptor selectivity, cyclization also imposes constraints that enhance conformational homogeneity and facilitates conformational analysis. The common modes of cyclization are the same found in naturally occurring cyclic peptides. These include side chain to side chain cyclization or side chain to end-group cyclization. For this purpose, amino acid side chains that are not involved in receptor recognition are connected together or to the peptide backbone. Another common cyclization is the end-to-end cyclization.
Three representative examples are compounds wherein partial structures of each peptide are made into rings by linking two penicillamine residues with a disulfide bridge (Mosberg et al., P.N.A.S. US, 80:5871, 1983), by formation of an amide bond between a lysine and an aspartate group (Charpentier et al., J. Med. Chem. 32:1184, 1989), or by connecting two lysine groups with a succinate unit (Rodriguez et al., Int. J. Pept. Protein Res. 35:441, 1990). These structures have been disclosed in the literature in the case of a cyclic enkephalin analog with selectivity for the xcex4-opiate receptor (Mosberg et al., ibid.); or as agonists to the cholecystokinin B receptor, found largely in the brain (Charpentier et al., ibid., Rodriguez et al., ibid.).
The main limitations to these classical modes of cyclization are that they require substitution of amino acid side chains in order to achieve cyclization.
Another conceptual approach to the conformational constraint of peptides was introduced by Gilon, et al., (Biopolymers, 31:745, 1991) who proposed backbone to backbone cyclization of peptides. The theoretical advantages of this strategy include the ability to effect cyclization via the carbons or nitrogens of the peptide backbone without interfering with side chains that may be crucial for interaction with the specific receptor of a given peptide. While the concept was envisaged as being applicable to any linear peptide of interest, in point of fact the limiting factor in the proposed scheme was the availability of suitable building units that must be used to replace the amino acids that are to be linked via bridging groups. The actual reduction to practice of this concept of backbone cyclization was prevented by the inability to devise any practical method of preparing building units of amino acids other than glycine (Byk et al., J. Org. Chem., 587:5687, 1992). While analogs of other amino acids were attempted the synthetic method used was unsuccessful or of such low yield as to preclude any general applicability.
In Gilon, EPO Application No. 564,739 A2; and J. Org. Chem., 57:5687, 1992, two basic approaches to the synthesis of building units are described. The first starts with the reaction of a diamine with a general a bromo acid. Selective protection of the w amine and further elaborations of protecting groups provides a building unit, suitable for Boc chemistry peptide synthesis. The second approach starts with selective protection of a diamine and reaction of the product with chloroacetic acid to provide the protected glycine derivative, suitable for Fmoc peptide synthesis.
Both examples deal with the reaction of a molecule of the general type Xxe2x80x94CH(R)xe2x80x94COxe2x80x94ORxe2x80x2 (wherein X represents a leaving group which, in the examples given, is either Br or Cl) with an amine which replaces the X. The amine bears an alkylidene chain which is terminated by another functional group, amine in the examples described, which may or may not be blocked by a protecting group.
In all cases the a nitrogen of the end product originates in the molecule which becomes the bridging chain for subsequent cyclization. This approach was chosen in order to take advantage of the higher susceptibility to nucleophilic displacement of a leaving group next to a carboxylic group.
In a molecule where R is different than hydrogen there is a high tendency to eliminate HX under basic conditions. This side reaction reduces the yield of Gilon""s method to the point where it is impractical for production of building units based on amino acids other than glycine. The diamine nitrogen is primary while the product contains a secondary nitrogen, which is a better nucleophile. So while the desired reaction may be sluggish, and require the addition of catalysts, the product may be contaminated with double alkylation products. There is no mention of building units with end group chemistries other than nitrogen, so the only cyclization schemes possible are backbone to side chain and backbone to C terminus.
Applications of Conformationally Constrained Peptides
Conformationally constrained peptides find many pharmacological uses. Somatostatin is a cyclic tetradecapeptide found both in the central nervous system and in peripheral tissues. It was originally isolated from mammalian hypothalamus and identified as an important inhibitor of growth hormone secretion from the anterior pituitary. Its multiple biological activities include inhibition of the secretion of glucagon and insulin from the pancreas, regulation of most gut hormones and regulation of the release of other neurotransmitters involved in motor activity and cognitive processes throughout the central nervous system (for review see Lamberts, Endocrine Rev., 9:427, 1988).
Natural somatostatin (also known as Somatotropin Release Inhibiting Factor, SRIF) of the following structure:
H-Ala1-Gly2-Cys3-Lys4-Asn5-Phe6-Phe7-Trp8-Lys9-Thr10-Phe11-Thr12-Ser13-Cys14-OH
was first isolated by Guillemin and colleagues (Brazeau et al. Science, 179:78, 1973). In its natural form, it has limited use as a therapeutic agent since it exhibits two undesirable properties: poor bioavailability and short duration of action. For this reason, great efforts have been made during the last two decades to find somatostatin analogs that will have superiority in either potency, biostability, duration of action or selectivity with regard to inhibition of the release of growth hormone, insulin or glucagon.
Structure-activity relation studies, spectroscopic techniques such as circular dichroism and nuclear magnetic resonance, and molecular modeling approaches reveal the following: the conformation of the cyclic part of natural somatostatin is most likely to be an antiparallel xcex2-sheet; Phe6 and Phe11 play an important role in stabilizing the pharmacophore conformation through hydrophobic interactions between the two aromatic rings; the four amino acids Phe7-Trp8-Lys9-Thr10 which are spread around the xcex2-turn in the antiparallel xcex2-sheet are essential for the pharmacophore; and (D)Trp8 is almost always preferable to (L)Trp8.
Nevertheless, a hexapeptide somatostatin analog containing these four amino acids anchored by a disulfide bridge: 
is almost inactive both in vitro and in vivo, although it has the advantage of the covalent disulfide bridge which replaces the Phe6-Phe11 hydrophobic interactions in natural somatostatin.
Four main approaches have been attempted in order to increase the activity of this hexapeptide somatostatin analog. (1) Replacing the disulfide bridge by a cyclization which encourages a cis-amide bond, or by performing a second cyclization to the molecule yielding a bicyclic analog. In both cases the resultant analog has a reduced number of conformational degrees of freedom. (2) Replacing the original amino acids in the sequence Phe7-(D)Trp8-Lys9-Thr10 with more potent amino acid analogs, such as replacing Phe7 with Tyr7 and Thr10 with Val10. (3) Incorporating additional structural elements from natural somatostatin with the intention that these new elements will contribute to the interaction with the receptor. (4) Eliminating one of the four amino acids Phe7-(D)Trp8-Lys9-Thr10 with the assumption that such analogs would be more selective.
The somatostatin analog, MK-678:
cyclo(N-Me-Ala7-Tyr7-(D)Trp8-Lys9-Val10-Phe)
is an example of a highly potent somatostatin analog designed using the first three approaches above (Lymangrover, et al., Life Science, 34:371, 1984). In this hexapeptide analog, a cis-amide bond is located between N-Me-Ala and Phe11, Tyr7 and Val10 replace Phe7 and Thr10 respectively, and Phe11 is incorporated from natural somatostatin.
Another group of somatostatin analogs (U.S. Pat. Nos. 4,310,518 and 4,235,886) includes octreotide: 
the only somatostatin analog currently available. It was developed using the third approach described above. Here, (D)Phe5 and the reduced C-terminal Thr12-CH2OH are assumed to occupy some of the conformational space available to the natural Phe6 and Thr12, respectively.
The compound TT2-32: 
is closely related to octreotide and is an example of implementing the fourth approach described above. The lack of Thr10 is probably responsible for its high selectivity in terms of antitumor activity.
These examples of highly potent somatostatin analogs indicate that the phenylalanines in positions 6 and 11 not only play an important role in stabilizing the pharmacophore conformation but also have a functional role in the interaction with the receptor. It is still an open question whether one phenylalanine (either Phe6 or Phe11) is sufficient for the interaction with the receptor or whether both are needed.
It is now known that the somatostatin receptors constitute a family of five different receptor subtypes (Bell and Reisine, Trends Neurosci., 16, 34-38, 1993), which may be distinguished on the basis of their tissue specificity and/or biological activity. Somatostatin analogs known in the art may not provide sufficient selectivity or receptor subtype selectivity, particularly as anti-neoplastic agents (Reubi and Laissue, TIPS, 16, 110-115, 1995).
Symptoms associated with metastatic carcinoid tumors (flushing and diarrhea) and vasoactive intestinal peptide (VIP) secreting adenomas (watery diarrhea) are treated with somatostatin analogs. Somatostatin has been also approved for the treatment of severe gastrointestinal hemorrhages. Somatostatin may also be useful in the palliative treatment of other hormone-secreting tumors (e.g., pancreatic islet-cell tumors and acromegaly) and hormone dependent tumors (e.g., chondrosarcoma and osteosarcoma) due to its anti-secretory activity.
Another important peptide, Bradykinin, is a naturally occurring nonapeptide,
Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9,
formed and released from precursors in the blood in response to inflammatory stimuli. Elevated levels of bradykinin also appear in other body fluids and tissues in pathological states such as asthma, septic shock and common cold. No clinical abnormalities have been associated so far with bradykinin deficiency which indicates that bradykinin may not play a critical role in normal physiology.
However, bradykinin mediates its physiological activities by binding to a specific receptive molecule called the bradykinin receptor. Two such bradykinin receptors have been identified so far (these are called B1 and B2 receptors). Subsequent to binding, the bradykinin signal transduction pathway includes production of prostaglandins and leukotrienes as well as calcium activation. Through these mediators, bradykinin is involved in pain, inflammation, allergic reactions and hypotension. Therefore, a substance that can block the ability of bradykinin to bind to its receptor, namely a bradykinin antagonist, should have a significant therapeutic value for one of the following indications: asthma, inflammation, septic shock, pain, hypotension and allergy.
The analog used herein to exemplify backbone cyclization is:
D-Arg0-Arg-R1-Hyp3-Gly-Phe-R2-D-Phe-Phe7-Arg
(wherein, R1 is Pro, R2 is Ser in native bradykinin). The change of proline at position 7 of native bradykinin to D-Phe confers antagonist activity. This compound was described in Steranka, et al., P.N.A.S. U.S., 85:3245-3249, 1988 and is one of a plethora of candidate sequences for modification by the current technology, i.e. backbone cyclization. In this regard, it is worth noting the applications: WO89/01781, EP-A-0370453 and EP-A-0334244 which disclose a widerange of candidate structures. Antagonist peptides on which stability and/or tissue selectivity can be conferred by appropriate cyclization will be selected from the many such known sequences.
According to the present invention a novel synthetic approach is disclosed providing Nxcex1(xcfx89(functionalized)alkylene)amino acid building units that can be used to synthesize novel Nxcex1-backbone cyclized peptide analogs such as, but not limited to, novel somatostatin and bradykinin analogs. None of the above-mentioned references teaches or suggests Nxcex1-(xcfx89(functionalized)alkylene)amino acids or the novel Nxcex1-backbone cyclized peptide analogs of the present invention.
It is an object of the present invention to provide backbone cyclized peptide analogs that comprise peptide sequences which incorporate at least two building units, each of which contains one nitrogen atom of the peptide backbone connected to a bridging group as described below. In the present invention, one or more pairs of the building units is joined together to form a cyclic structure. Thus, according to one aspect of the present invention, backbone cyclized peptide analogs are provided that have the general Formula (I): 
wherein: a and b each independently designates an integer from 1 to 8 or zero; d, e, and f each independently designates an integer from 1 to 10; (AA) designates an amino acid residue wherein the amino acid residues in each chain may be the same or different; E represents a hydroxyl group, a carboxyl protecting group or an amino group, or COxe2x80x94E can be reduced to CH2xe2x80x94OH; R. Rxe2x80x2, Rxe2x80x3, and Rxe2x80x2xe2x80x3 each designates an amino acid side-chain such as H, CH3, etc., optionally bound with a specific protecting group; and the lines independently designate a bridging group of the Formula: (i) xe2x80x94Xxe2x80x94Mxe2x80x94Yxe2x80x94Wxe2x80x94Zxe2x80x94 ; or (ii) xe2x80x94Xxe2x80x94Mxe2x80x94Zxe2x80x94 wherein: one line may be absent; M and W are independently selected from the group consisting of disulfide, amide, thioether, thioesters, imines, ethers and alkenes; and X, Y and Z are each independently selected from the group consisting of alkylene, substituted alkylene, arylene, homo- or hetero-cycloalkylene and substituted cycloalkylene.
In certain preferred embodiments, the COxe2x80x94E group of Formula (I) is reduced to a CH2OH group.
Another embodiment of the present invention involves N-backbone to side chain cyclized peptides of the general formula (II): 
wherein the substituents are as defined above.
A preferred embodiment of the present invention involves the backbone cyclized peptide analog of Formulae I or II wherein the line designates a bridging group of the Formula: xe2x80x94(CH2)xxe2x80x94Mxe2x80x94(CH2)yxe2x80x94Wxe2x80x94(CH2)zxe2x80x94 wherein M and W are independently selected from the group consisting of disulfide, amide, thioether, thioesters, imines, ethers and alkenes; x and z each independently designates an integer from 1 to 10, and y is zero or an integer of from 1 to 8, with the proviso that if y is zero, W is absent.
Further preferred are backbone cyclized peptide analogs of the Formula I or II wherein R and Rxe2x80x2 are other than H, such as CH3, (CH3)2CHxe2x80x94, (CH3)2CHCH2xe2x80x94, CH3CH2CH(CH3)xe2x80x94, CH3S(CH2)2xe2x80x94, HOCH2xe2x80x94, CH3CH(OH)xe2x80x94, HSCH2xe2x80x94, NH2C(xe2x95x90O)CH2xe2x80x94, NH2C(xe2x95x90O)(CH2)2xe2x80x94, NH2(CH2)3xe2x80x94, HOC(xe2x95x90O)CH2xe2x80x94, HOC(xe2x95x90O)(CH2)2xe2x80x94, NH2(CH2)4xe2x80x94, C(NH2)2 NH(CH2)3xe2x80x94, HO-phenyl-CH2xe2x80x94, benzyl, methylindole, and methylimidazole.
A more preferred embodiment of the present invention is directed to backbone cyclization to stabilize the xcex2-turn conformation of bradykinin analogs of the general Formula (III): 
wherein M is an amide bond, x and z are each independently an integer of 1 to 10, and K is H or an acyl group.
Also more preferred are backbone cyclized peptide analogs of the present invention comprising bradykinin analogs of the general Formula (IVa): 
wherein M is an amide bond, x and z are each independently an integer of 1 to 10, K is H or an acyl group, and R6 is Gly or Ser; or the general Formula (IVb): 
wherein x is an integer of 1 to 10; K is H or an acyl group; (R6) is selected from the group of D-Asp, L-Asp, D-Glu and L-Glu; and z is according to the amino acid specified: 1 in case of D and L-Asp, and 2 in the case of D and L Glu.
Further more preferred backbone cyclized peptide analogs according to the present invention having bradykinin antagonist activity have the Formula (V): 
wherein M is an amide bond, x and z are each independently an integer of 1 to 10, and K is H or an acyl group.
Specifically preferred backbone cyclized peptide analogs of the present invention are:
1) Ada-(D)Arg-Arg-cyclo(Nxcex1(1-(6-aminohexylene)Gly-Hyp-Phe-D-Asp)-D-Phe-Phe-Arg-OH;
2) H-D-Arg-Arg-cyclo(Nxcex1(1-(4-propanoyl))Gly-Hyp-Phe-Nxcex1(3-amido-propylene)Gly)-Ser-D-Phe-Phe-Arg-OH; and
3) H-D-Arg-Arg-cyclo(Nxcex1(4-propanoyl)Gly-Hyp-Phe-N(3-amido-propyl)-S-Phe)-Ser-D-Phe-Phe-Arg-OH.
Another preferred aspect of the present invention is directed to backbone cyclizatiog to generate novel somatostatin analogs linked between positions 6 and 11, leaving the phenylalanine side chains untouched. This conformational stabilization is much more rigid than the Phe6, Phe11 hydrophobic interaction in natural somatostatin and is more stable to reduction/oxidation reactions than the Cys-Cys disulfide bridge. In other words, for the first time a stable covalent bridge can be achieved while either one or both of the original Phe6 and Phe11 are retained.
Moreover, backbone cyclizations can also be used to anchor the xcex2-turn, not only in positions 6 and 11 but also inside the active reaction of Phe7-(D)Trp8-Lys9-Thr10, yielding either a monocyclic analog with a preferable conformation or a very rigid bicyclic analog. Here again, the side chains of the pharmacologically active amino acids remain untouched and the only change is in limiting the conformational space.
As used herein and in the claims in the following more preferred backbone cyclized peptide analogs, the superscript numbers following the amino acids refer to their position numbers in the native Somatostatin.
A more preferred backbone cyclized peptide novel analog is the Formula (XIVa): 
with a most preferred analog being the Formula (XIVb): 
wherein m and n are 1, 2 or 3; X is CH2OH or CONH2; R5 is absent or is Gly, (D)- or (L)-Ala, Phe, Nal and xcex2-Asp(Ind); R6 and R11 are independently Gly or (D)- or (L)-Phe; R7 is Phe or Tyr; R10 is absent or is Gly, Abu, Thr or Val; R12 is absent or is Thr or Nal, and Y2 is selected from the group consisting of amide, disulfide, thioether, imines, ethers and alkenes. In these monocyclic somatostatin analogs, a backbone cyclization replaces the Cys6-Cys11 disulfide bridge, leaving the phenylalanine side chains as in the natural somatostatin. Still more preferred is the analog wherein Phe7 is replaced with Tyr7 and Thr10 is replaced with Val10.
Other more preferred monocyclic analogs that anchor the molecule in positions inside the active region rather than in positions 6 and 11 are formulae XV (a and b) and XVI (a-c): 
wherein i and j are independently 1, 2 or 3; X is CH2OH or CONH2; R5 is absent or is (D)- or (L)-Phe, Nal, or xcex2-Asp(Ind); R6 is (D) or (L)-Phe; R10 is absent or is Gly, Abu or Thr; and R11 is (D)- or (L)-Phe; R12 is absent or is Thr or Nal, and Y1 is selected from the group consisting of amide, disulfide, thioether, imines, ethers and alkenes.
Still other more preferred analogs incorporate backbone cyclization in positions 6 and 11 as in Formula XIV, together with the backbone cyclizations as in Formula XV and XVI, yielding rigid bicyclic analogs of the Formulae XVII (a and b) and XVIII (a and b): 
wherein i, j, m and n are independently 1, 2 or 3; X is CH2OH or CONH2; R5 is absent or is (D)- or (L)-Phe, Nal, or xcex2-Asp(Ind); R6 and R11 are independently Gly or (D)- or (L)-Phe; R10 is absent or is Gly, Abu, Val or Thr; R12 is absent or is Thr or Nal; and Y1 and Y2 are independently selected from the group consisting of amide, disulfide, thioether, imines, ethers and alkenes.
Other more preferred bicyclic analogs differ from Formulae XVII and XVIII by the replacement of the amino acids at positions 6 and 11 by cysteines which form a disulfide bond, leaving only one backbone cyclization in the Formulae XIX (a and b) and XX (a and b): 
wherein i and j are independently 1, 2 or 3; X is CH2OH or NH2; R5 is absent or is (D)- or (L)-Phe, Nal, or xcex2-Asp(Ind); R6 and R11 are independently Gly or Phe; R10 is absent or is Gly, Abu or Thr; R12 is absent or is Thr or Nal; and Y1 is selected from the group consisting of amide, disulfide, thioether, imines, ethers and alkenes.
Another aspect of the present invention is a method for the preparation of cyclic peptides of the general Formula (I): 
wherein: a and b each independently designates an integer from 1 to 8 or zero; d, e, and f each independently designates an integer from 1 to 10; (AA) designates an amino acid residue wherein the amino acid residues in each chain may be the same or different; E represents a hydroxyl group, a carboxyl protecting group or an amino group, or COxe2x80x94E can be reduced to CH2xe2x80x94OH; R, Rxe2x80x2, Rxe2x80x3, and Rxe2x80x2xe2x80x3 each designates an amino acid side-chain optionally bound with a specific protecting group; and the lines designate a bridging group of the Formula:
(i) xe2x80x94Xxe2x80x94Mxe2x80x94Yxe2x80x94Wxe2x80x94Zxe2x80x94; or (ii) xe2x80x94Xxe2x80x94Mxe2x80x94Zxe2x80x94
wherein: one line may be absent; M and W are independently selected from the group consisting of disulfide, amide, thioether, thioesters, imines, ethers and alkenes; and X, Y and Z are each independently selected from the group consisting of alkylene, substituted alkylene, arylene, homo- or hetero-cycloalkylene and substituted cycloalkylene. This method comprises the steps of incorporating at least one Nxcex1-xcfx89-functionalized derivative of amino acids of Formula (VI): 
wherein X is a spacer group selected from the group consisting of alkylene, substituted alkylene, arylene, cycloalkylene and substituted cycloalkylene; Rxe2x80x2 is an amino acid side chain, optionally bound with a specific protecting group; B is a protecting group selected from the group consisting of alkyloxy, substituted alkyloxy, or aryl carbonyls; and G is a functional group selected from the group consisting of amines, thiols, alcohols, carboxylic acids and esters, aldehydes, alcohols and alkyl halides; and A is a specific protecting group of G; into a peptide sequence and subsequently selectively cyclizing the functional group with one of the side chains of the amino acids in said peptide sequence or with another xcfx89-functionalized amino acid derivative.
A further object of the present invention is directed to building units known as a Nxcex1-xcfx89-functionalized derivatives of the general Formula (VI) of amino acids which are prerequisites for the cyclization process: 
wherein X is a spacer group selected from the group consisting of alkylene, substituted alkylene, arylene, cycloalkylene and substituted cycloalkylene; R is the side chain of an amino acid, optionally bound with a specific protecting group; B is a protecting group selected from the group consisting of alkyloxy, substituted alkyloxy, or aryloxy carbonyls; and G is a functional group selected from the group consisting of amines, thiols, alcohols, carboxylic acids and esters, aldehydes and alkyl halides; and A is a protecting group thereof.
Preferred building units are the xcfx89-functionalized amino acid derivatives wherein X is alkylene; G is a thiol group, an amine group or a carboxyl group; R is phenyl, methyl or isobutyl; with the proviso that when G is an amine group, R is other than H.
Further preferred are xcfx89-functionalized amino acid derivatives wherein R is protected with a specific protecting group.
More preferred are xcfx89-functionalized amino acid derivatives of the Formulae: 
wherein X, R, A and B are as defined above.
Specifically preferred xcfx89-functionalized amino acid derivatives include the following:
1) Nxcex1-(Fmoc) (3-Boc-amino propylene)-(S)Phenylalanine;
2) Nxcex1-(Fmoc) (3-Boc-amino propylene)-(R)Phenylalanine;
3) Nxcex1-(Fmoc) (4-Boc-amino butylene)-(S)Phenylalanine;
4) Nxcex1-(Fmoc) (3-Boc-amino propylene)-(S)Alanine;
5) Nxcex1-(Fmoc) (6-Boc-amino hexylene)-(S)Alanine;
6) Nxcex1-(Fmoc) (3-Boc-amino propylene)-(R)Alanine;
7) Nxcex1-(2-(benzylthio)ethylene)glycine ethyl ester;
8) Nxcex1-(2-(benzylthio)ethylene)(S)leucine methyl ester;
9) Nxcex1-(3-(benzylthio)propylene)(S)leucine methyl ester:
10) Boc-Nxcex1-(2-(benzylthio)ethylene)glycine;
11) Boc-Nxcex1-(2-(benzylthio)ethylene)(S)phenylalanine;
12) Boc-Nxcex1-(3-(benzylthio)propylene)(S)phenylalanine;
13) Boc-L-phenylalanyl-Nxcex1-(2-(benzylthio)ethylene) glycine ethyl ester;
14) Boc-L-phenylalanyl-Nxcex1-(2-(benzylthio)ethylene)-(S)phenylalanine methyl ester;
15) Nxcex1(Fmoc)-(2-t-butyl carboxy ethylene)glycine;
16) Nxcex1(Fmoc)-(3-t-butyl carboxy propylene)glycine;
17) Nxcex1(Fmoc) (2-t-butyl carboxy ethylene)(S)phenylalanine;
18) Nxcex1(Fmoc) (2-Boc amino ethylene)glycine;
19) Nxcex1(Fmoc) (3-Boc amino propylene)glycine;
20) Nxcex1(Fmoc) (4-Boc amino butylene)glycine; and
21) Nxcex1(Fmoc) (6-Boc amino hexylene)glycine.
Novel, practical, generally applicable processes for the preparation of these Nxcex1-xcfx89-functionalized derivatives of amino acids are a further aspect of this invention.
As such, an object of this invention is a method of making an xcfx89-functionalized amino acid derivative of the general Formula: 
wherein X is a spacer group selected from the group consisting of alkylene, substituted alkylene, arylene, cycloalkylene and substituted cycloalkylene; R is the side chain of an amino acid, such as H, CH3, etc.; A and B are protecting groups selected from the group consisting of alkyloxy, substituted alkyloxy, or aryloxy carbonyls;
comprising the steps of:
i) reacting a diamine compound of the general Formula: 
wherein A, B and X are as defined above,
with a triflate of Formula CF3SO2xe2x80x94Oxe2x80x94CH(R)xe2x80x94COxe2x80x94E wherein E is a carboxyl protecting group and R is as defined above; to yield a compound of Formula: 
wherein A, B, E, R and X are as defined above
ii) and deprotecting the carboxyl to yield an Nxcex1 xcfx89-functionalized amino acid derivative, wherein the xcfx89-functional group is an amine.
A further object of this invention is a method of making an xcfx89-functionalized amino acid derivative of the general Formula: 
where B is a protecting group selected from the group of substituted alkyloxy, substituted alkyloxy, or aryloxy carbonyls; R is the side chain of an amino acid, such as H, CH3, etc.; X is a spacer group selected from the group of alkylene, substituted alkylene, arylene, cycloalkylene or substituted cycloalkylene; and A is a protecting group selected from the group of alkyl or substituted alkyl, thio ethers or aryl or substituted aryl thio ethers;
comprising the steps of:
i) reacting a compound of the general Formula Bxe2x80x94NHxe2x80x94Xxe2x80x94Sxe2x80x94A with a triflate of the general Formula CF3SO2xe2x80x94Oxe2x80x94CH(R)xe2x80x94COxe2x80x94E wherein E is a carboxyl protecting group and A, X and R are as defined above, to give a compound of the Formula: 
ii) selectively removing the protecting group E, and
iii) protecting the free amino group to yield an Nxcex1(xcfx89-functionalized) amino acid derivative, wherein the xcfx89-functional group is a thiol.
A further object of this invention is a method of making an xcfx89-functionalized amino acid derivative of the general Formula: 
where B is a protecting group selected from the group of alkyloxy, substituted alkyloxy, or aryloxy carbonyls; R is the side chain of an amino acid, such as H, CH3, etc.; X is a spacer group selected from the group of alkylene, substituted alkylene, arylene, cycloalkylene or substituted cycloalkylene; and A is a protecting group selected from the group of alkyl or substituted alkyl, esters, or thio esters or substituted aryl esters or thio esters;
comprising the steps of:
i) reacting a compound of the general Formula Bxe2x80x94NHxe2x80x94Xxe2x80x94COxe2x80x94A with a triflate of the general Formula CF3SO2xe2x80x94Oxe2x80x94CH(R)xe2x80x94COxe2x80x94E wherein E is a carboxyl protecting group and A, B, X and R are as defined above, to give a compound of Formula: 
ii) and selectively removing protecting group E, to yield an Nxcex1(xcfx89-functionalized) amino acid derivative, wherein the xcfx89-functional group is a carboxyl.
A further aspect of this invention is to provide methods for the preparation of novel backbone cyclic peptides, comprising the steps of incorporating at least one Nxcex1-xcfx89-functionalized derivatives of amino acids into a peptide sequence and subsequently selectively cyclizing the functional group with one of the side chains of the amino acids in said peptide sequence, or with another xcfx89-functionalized amino acid derivative.
Backbone cyclized analogs of the present invention may be used as pharmaceutical compositions and for methods for the treatment of disorders including: acute asthma, septic shock, brain trauma and other traumatic injury, post-surgical pain, all types of inflammation, cancers, endocrine disorders and gastrointestinal disorders.
Therefore, further objects of the present invention are directed to pharmaceutical compositions comprising pharmacologically active backbone cyclized peptide agonists and antagonists prepared according to the methods disclosed herein and a pharmaceutically acceptable carrier or diluent; and methods for the treatment of inflammation, septic shock, cancer or endocrine disorders and gastrointestinal disorders therewith.